The overall aim of the project is to identify the protein(s) that mediate(s) Na+-H+ exchange across the luminal membrane of the proximal tubule cell, and to clone and sequence the structural gene for this transport system. Previous studies in the sponsor's laboratory employing covalent labeling of rabbit renal microvillus membranes with dicyclohexylcarbodiimide and methylisobutylamiloride have identified at least two different polypeptides as candidates for the Na+-H+ exchanger or one of its subunits. The co-sponsor's laboratory recently isolated mutant lines of LLC-PK1 kidney cells that are either deficient in or overexpress Na+-H+ exchange activity. In the proposed studies, covalent labeling of apical membrane vesicles isolated from these mutant cell lines will be performed to define which of the previously identified candidate polypeptides actually represents the Na+-H+ exchanger or one of its subunits. Monoclonal antibodies to this polypeptide will then be generated. Specificity of the antibodies will be confirmed by use of a reconstitution assay for Na+-H+ exchange activity available in the sponsor's laboratory. These antibodies will then be used to affinity purify the transport protein. A lambda gtll cDNA expression library previously prepared in the sponsor's laboratory from rabbit renal cortex will then be screened with antibodies against the transport protein or with oligonucleotide probes constructed on the basis of partial sequencing of peptides derived from the purified protein. Isolated cloned cDNA's will be sequenced and the primary structure of the protein thereby deduced. Subsequent studies will address the secondary structure, mechanism of action, biosynthesis, and regulation of the Na+-H+ exchanger. Because the plasma membrane Na+-H+ exchanger is a ubiquitous transport system involved in diverse aspects of cell physiology, the proposed studies will be of general biomedical significance. Most importantly, the project will provide training in a variety of techniques relevant to membrane physiology and molecular biology including tissue culture, membrane isolation, generation of monoclonal antibodies, protein biochemistry, and recombinant DNA technology.